1. Field of the Invention
The present invention relates to methods and compositions for initiating, promoting and directing cell attachment, migration and cell to cell assembly in response to multimerization of ligands and/or receptors. In particular, the present invention provides multimeric ligands which bind receptors of the Eph receptor tyrosine kinase family to selectively modulate specific cell activities such as cell attachment and cell to cell assembly by promoting the formation of multimeric receptors of specific composition.
2. Background Art
Developmental organization and subsequent remodeling of vasculature obligates vascular endothelial cells and their progenitors to use cell-cell recognition and targeting machinery, both in the initial stages of vasculogenesis and in the angiogenic remodeling required for organogenesis, wound healing and tumor growth. Similar to the developing neural system, vascular cells must migrate, discriminate and assemble with appropriate partner cells to establish and remodel highly integrated and interconnected cellular networks. Early developmental xe2x80x9cvasculogenicxe2x80x9d assembly of blood vessels requires that endothelial progenitor cells migrate in response to yet unidentified cues, then discriminate among cells they contact to assemble vascular structures with similarly programmed endothelial progenitors (32). During the vascular remodeling occurring during organogenesis, wound healing and tumor growth, endothelial cells in existing vessels receive angiogenic cues to invade their basement membrane and sprout long migrating cellular processes that resemble axons (33,34). Cultured microvascular endothelial cells extend similar processes that track along fibrillar strands of connective tissue matrix to contact and engage appropriate partner cells to form cord and tube structures during in vitro capillary-like morphogenesis. In vivo (and in vitro), these processes encounter and discriminate among cells until appropriate partners for assembly are identified. On engagement of processes extended from existing, efferent limb vessels, specialized interendothelial cellular connections are established to ultimately interconnect lumen (35). This entire scenario is quite similar to the axonal extension, migration, discrimination and targeting processes that direct correct assembly of neural networks.
An enlarging body of evidence has assigned members of the Eph family of receptor tyrosine kinases crucial roles in neural targeting, and early data also support their function in the assembly of vascular structures (1,2,3,7,25). As a class, the Eph family of receptors and their ligands are tissue restricted in their distribution and are highly diverse, with over 13 distinct receptors interacting with distinct ligands. During development, specific Eph family receptors are expressed in distinct tissue sites that are bounded by adjacent tissues expressing their membrane-bound ligands in a reciprocal compartmentation pattern (26). A topographically defined gradient of one ligand, ephrin-A2 (ELF-1), on membranes in the tectum directs the correct targeting of retinal axons that express differential levels of its receptor, EphA3 (Mek 4) (36). Interestingly, regional overexpression of ephrin-A2 (ELF-1) misdirects these projections (37). These and other experiments emphasize the important role these Eph family receptors and ligands play in directing neuronal cell-cell interactions in developing nervous tissue.
Eph family receptors are subdivided into two functional classes by their affinities for membrane-bound ligands of two different structural types. Receptors of the EphA subfamily, including EphA3 (Mek 4), EphA5 (Ehk-1) and others, bind ligands that are membrane-associated through glycerophosphatidylinositol (GPI) linkages, and may be released by phospholipases C and D (41). The GPI-linked ligands characterized to date are ephrin-A1, ephrin-A3, ephrin-A4, ephrin-A2 and ephrin-A5 (formerly called LERKs 1,3,4,6,and 7) (2,7,36,38). The EphB receptor subfamily members show overlapping high affinities for ligands that are transmembrane proteins, including ephrin-B1, ephrin-B2 and ephrin-B3 (formerly called LERKs 2,5 and 8) (9,10,30,39). The transmembrane spanning ligands show remarkable amino acid conservation on the carboxy terminus, implying conservation of structure important in their function, and clouding the distinction between receptors and ligands.
Recent evidence suggests the capacity of these xe2x80x9cligandsxe2x80x9d to signal through engagement of xe2x80x9creceptorxe2x80x9d extracellular domains. Engagement of ephrin-B1 (xe2x80x9cligandxe2x80x9d) by recombinant EphB2 (Nuk) (xe2x80x9creceptorxe2x80x9d) ectodomain initiates tyrosine phosphorylation of ephrin-B1 (28). Moreover, guidance of hippocampal neurons appears directed by their expression of xe2x80x9cligandsxe2x80x9d for EphB2 (Nuk), a xe2x80x9creceptorxe2x80x9d ectodomain they engage during the course of decussation through the anterior commissure during development (23). Thus, bidirectional signaling may be initiated by juxtacrine ligand-receptor engagement on cell-cell contact. Such bidirectional signaling is attractive as an intermediate step in cell-cell recognition and commitment to assemble multicellular structures.
Both EphA2 (Eck) and EphB1 (ELK) have been implicated as important intermediaries during angiogenesis. The primary ligand for EphA2 (Eck) was first cloned as a TNFxcex1-induced product of cultured human umbilical vein endothelial cells (ephrin-A1, previously called B-61 or LERK-1) (39,40). Ephrin-A1 is a GPI-linked membrane protein that is also released into a soluble fraction where it may promote migration of bovine endothelial cells through its interaction with EphA2 (Eck) (7). Antibodies against ephrin-A1 interrupt TNFxcex1-induced angiogenic responses in the rat cornea assay, consistent with their interruption of ephrin-A1 to promote angiogenesis through EphA2 (Eck) (7).
EphB1 (ELK) and ephrin-B1 (LERK-2) are both expressed on mesenchymal progenitors of vascular cells, on glomerular capillary endothelial cells in mature kidney and on human umbilical vein endothelial cells (8). The early expression of ephrin-B1 and EphB1 in renal glomerular microvascular progenitor cells has suggested their participation in targeting and capillary assembly in this specialized microcirculation (8). Ephrin-B1 promotes assembly of human renal microvascular endothelial cells (HRMEC) into capillary-like structures (8), yet human umbilical vein endothelial cells (HUVEC) are not responsive, despite their expression of EphB1, and its tyrosine phosphorylation in response to ephrin-B1. In contrast, ephrin-A 1 (LERK-1) has no capillary-assembly activity for HRMEC, yet promotes capillary-like assembly of HUVEC (8). Thus, the downstream signaling responses in vascular endothelial cells from different microcirculations distinguish among Eph receptor ligands to determine different cellular responses.
The present invention provides methods for selectively modulating cell attachment, cell migration, cell to cell assembly and other activities regulated by Eph receptor activation through the promotion or inhibition of multimerization of the receptors which regulate these functions by signal transduction.
The present invention provides a method for initiating, promoting and/or directing cell attachment to a matrix or to another cell, comprising contacting an EphB receptor-expressing cell with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby initiating, promoting and/or directing cell attachment to a matrix or to another cell.
Also provided is a method for initiating and/or promoting cell migration, comprising contacting an EphB receptor-expressing cell with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby initiating and/or promoting cell migration.
A method for promoting endothelialization of a prosthesis is also provided, comprising contacting the prosthesis with EphB receptor-expressing endothelial cells with a tetrameric EphB receptor-binding ligand whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting endothelialization of the prosthesis.
In addition, the present invention provides a method for promoting expression of a molecule on the surface of an ephB receptor-expressing cell, wherein the expressed molecule affects leukocyte or platelet attachment and migration, comprising contacting the cell with a tetrameric Eph receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting expression of a molecule on the surface of the cell which affects leukocyte or platelet attachment and migration.
Further provided is a method for promoting function of a molecule on the surface of an ephB receptor-expressing cell, wherein the expressed molecule affects leukocyte or platelet attachment and migration, comprising contacting the cell with a tetrameric EphB receptor-binding ligand whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting function of a molecule on the surface of the cell which affects leukocyte or platelet attachment and migration.
The present invention also provides a method for promoting migration, survival and/or targeting of an EphB receptor-expressing neural cell, comprising contacting the cell with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting migration, survival and/or targeting of the neural cell.
The present invention additionally provides a method for targeting and/or promoting endothelial cell incorporation at a site of endothelial cell injury or new blood vessel formation in a subject, comprising contacting an EphB receptor-expressing endothelial cell at the site of endothelial cell injury or new blood vessel formation in the subject with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the receptor, thereby targeting and/or promoting endothelial cell incorporation at the site of endothelial cell injury or new blood vessel formation in the subject.
Furthermore, the present invention provides a method for inhibiting cell attachment to a matrix or to another cell, comprising contacting an EphB receptor-expressing cell, which is stimulated to attach to a matrix or to another cell upon binding a tetrameric Eph receptor-binding ligand, with a substance which prevents binding of the tetrameric ligand to the EphB receptor, thereby inhibiting cell attachment to a matrix or to another cell.
A method for inhibiting cell migration is additionally provided, comprising contacting an EphB receptor-expressing cell, which is stimulated to migrate upon binding a tetrameric EphB receptor-binding ligand, with a substance which prevents binding of the tetrameric ligand to the EphB receptor, thereby inhibiting cell migration.
Additionally provided is a method for inhibiting expression of a molecule on the surface of an EphB receptor-expressing cell, wherein the expressed molecule affects leukocyte or platelet attachment and migration, comprising contacting an EphB receptor-expressing cell, which is stimulated to express a surface molecule which affects leukocyte or platelet attachment and migration upon binding a tetrameric EphB receptor-binding ligand, with a substance which prevents binding of the tetrameric ligand, thereby inhibiting expression of a molecule on the surface of the cell which affects leukocyte or platelet attachment and migration.
The present invention also provides a method for inhibiting function of a molecule on the surface of an EphB receptor-expressing cell, wherein the molecule affects leukocyte or platelet attachment and migration, comprising contacting an EphB receptor-expressing cell, which is stimulated to promote function of a surface molecule which affects leukocyte or platelet attachment and migration upon binding a tetrameric EphB receptor-binding ligand, with a substance which prevents binding of the tetrameric ligand, thereby inhibiting function of a molecule on the surface of the cell which affects leukocyte or platelet attachment and migration.
The present invention additionally provides a method for inhibiting migration, survival and/or targeting of a neural cell which expresses an EphB receptor, comprising contacting an EphB receptor-expressing neural cell, which is stimulated to promote migration, survival or targeting of the neural cell upon binding a tetrameric EphB receptor-binding ligand, with a substance which prevents binding of the tetrameric ligand, thereby inhibiting migration, survival and/or targeting of the neural cell.
The present invention also provides a composition comprising an isolated tetrameric EphB receptor ligand, a composition comprising an isolated tetrameric EphB receptor and a composition comprising an isolated tetrameric EphB receptor ligand/tetrameric EphB receptor complex.
Furthermore, the present invention provides a method for screening an EphB receptor-binding ligand for the ability to initiate, promote and/or direct cell attachment to a matrix or to another cell when in multimeric form, comprising:
a) contacting a multimeric EphB receptor-binding ligand with an EphB receptor-expressing cell under conditions whereby the ligand can bind the receptor; and
b) detecting attachment of cells which have bound multimeric ligand as compared to attachment of cells which have not bound multimeric ligand, whereby attachment of cells which have bound multimeric ligand and no attachment of cells which have not bound multimeric ligand identifies an EphB receptor-binding ligand with the ability to initiate, promote and/or direct cell attachment to a matrix or to another cell when in multimeric form.
A method for screening an EphB receptor-binding ligand for the ability to promote cell migration when in multimeric form is additionally provided, comprising:
a) contacting a multimeric EphB receptor-binding ligand with an EphB receptor-expressing cell under conditions whereby the ligand can bind the receptor; and
b) detecting migration of cells which have bound multimeric ligand as compared to migration of cells which have not bound multimeric ligand, whereby migration of cells which have bound multimeric ligand and no migration of cells which have not bound multimeric ligand identifies an EphB receptor-binding ligand with the ability to promote cell migration when in multimeric form.
Also provided is a method for screening an EphB receptor-binding ligand for the ability to promote expression of a molecule on the surface of an EphB receptor-expressing cell, wherein the expressed molecule affects leukocyte or platelet attachment and migration, when in multimeric form, comprising:
a) contacting a multimeric EphB receptor-binding ligand with a cell which expresses an EphB receptor under conditions whereby the ligand can bind the receptor; and
b) detecting expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have bound multimeric ligand as compared to expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have not bound multimeric ligand, whereby expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have bound multimeric ligand and no expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have not bound multimeric ligand identifies a ligand with the ability to promote the expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells, when the ligand is in multimeric form.
The present invention further provides a method for screening an EphB receptor-binding ligand for the ability to promote function of a molecule on the surface of an EphB receptor-expressing cell, wherein the function of the molecule affects leukocyte or platelet attachment and migration, when in multimeric form, comprising:
a) contacting a multimeric EphB receptor-binding ligand with an EphB receptor-expressing cell under conditions whereby the ligand can bind the receptor; and
b) detecting function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have bound multimeric ligand as compared to function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have not bound multimeric ligand, whereby function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have bound multimeric ligand and no function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have not bound multimeric ligand identifies a ligand with the ability to promote function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells, when the ligand is in multimeric form.
The present invention also provides a method for screening an EphB receptor-binding ligand for the ability to promote migration, survival and/or targeting of a neural cell when in multimeric form, comprising:
a) contacting a multimeric EphB receptor-binding ligand with an EphB receptor-expressing neural cell under conditions whereby the ligand can bind the receptor; and
b) determining migration, survival and/or targeting of a neural cell which has bound multimeric ligand as compared to migration, survival or targeting of a neural cell which has not bound multimeric ligand, whereby migration, survival or targeting of a neural cell which has bound multimeric ligand and no migration, survival, or targeting of a neural cell which has not bound multimeric ligand identifies a ligand with the ability to promote migration, survival or targeting of a neural cell when in multimeric form.
Additionally provided is a method for screening an EphB receptor for the ability to initiate, promote and/or direct cell attachment to a matrix or to another cell when the receptor is in multimeric form, comprising:
a) producing a multimeric EphB receptor on the surface of a cell which expresses an EphB receptor; and
b) detecting attachment of cells with a multimeric EphB receptor as compared to attachment of cells without a multimeric EphB receptor, whereby attachment of cells with a multimeric EphB receptor and no attachment of cells without a multimeric EphB receptor identifies an EphB receptor with the ability to initiate, promote and/or direct cell attachment to a matrix or to another cell when in multimeric form.
Further provided is a method for screening an EphB receptor for the ability to promote cell migration when the receptor is in multimeric form, comprising:
a) producing a multimeric EphB receptor on the surface of a cell which expresses an EphB receptor; and
b) detecting migration of cells with a multimeric EphB receptor as compared to migration of cells without a multimeric EphB receptor, whereby migration of cells with a multimeric EphB receptor and no migration of cells without a multimeric EphB receptor identifies an EphB receptor with the ability to promote cell migration when in multimeric form.
The present invention also provides a method for screening an EphB receptor for the ability to promote expression of a molecule on the surface of an EphB receptor-expressing, wherein the expressed molecule affects leukocyte or platelet attachment and migration, when the receptor is in multimeric form, comprising:
a) producing a multimeric EphB receptor on the surface of a cell which expresses an EphB receptor; and
b) detecting expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells with a multimeric EphB receptor as compared to expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells without a multimeric EphB receptor, whereby expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells with a multimeric EphB receptor and no expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells without a multimeric EphB receptor identifies an EphB receptor with the ability to promote expression of a molecule on the surface of a cell which expresses an EphB receptor, wherein the expressed molecule affects leukocyte or platelet attachment and migration, when the EphB receptor is in multimeric form.
The present invention additionally provides a method for screening an Eph receptor for the ability to promote function of a molecule on the surface of an EphB receptor-expressing cell, wherein the function of the molecule affects leukocyte or platelet attachment and migration, when the receptor is in multimeric form, comprising:
a) producing a multimeric EphB receptor on the surface of a cell which expresses an EphB receptor; and
b) detecting function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells with a multimeric EphB receptor as compared to function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells without a multimeric EphB receptor, whereby function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells with a multimeric EphB receptor and no function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells without a multimeric EphB receptor identifies an EphB receptor with the ability to promote function of a molecule on the surface of a cell which expresses an EphB receptor, wherein the expressed molecule affects leukocyte or platelet attachment and migration, when the EphB receptor is in multimeric form.
A method for screening an EphB receptor for the ability to promote migration, survival and/or targeting of a neural cell when the receptor is in multimeric form is also provided, comprising:
a) producing a multimeric EphB receptor on the surface of a neural cell which expresses an EphB receptor; and
b) detecting migration, survival or targeting of neural cells with a multimeric EphB receptor as compared to migration, survival or targeting of neural cells without a multimeric EphB receptor, whereby migration, survival and/or targeting of neural cells with a multimeric EphB receptor and no migration, survival or targeting of neural cells without a multimeric EphB receptor identifies an EphB receptor with the ability to promote migration, survival and/or targeting of neural cells when in multimeric form.
In addition, the present invention provides a method for screening a substance for the ability to inhibit the binding of a multimeric EphB receptor-binding ligand to an EphB receptor comprising:
a) contacting the substance with a cell expressing an EphB receptor;
b) contacting the cell of step (a) with a multimeric EphB receptor-binding ligand under conditions whereby the multimeric ligand can bind the receptor; and
c) detecting the binding of the multimeric ligand to the receptor, whereby no binding of the multimeric ligand to the receptor identifies a substance with the ability to inhibit the binding of a multimeric EphB receptor-binding ligand to an EphB receptor.
Also provided herein is a method for promoting angiogenesis, comprising contacting EphB receptor-expressing cells which are associated with angiogenesis with a multimeric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting angiogenesis.
A method for disrupting angiogenesis is additionally provided, comprising contacting an EphB receptor-expressing cell which promotes angiogenesis upon binding a multimeric EphB receptor-binding ligand with a substance which prevents formation of a multimeric EphB receptor-binding ligand or inhibits binding of the multimeric EphB receptor-binding ligand to the receptor, thereby disrupting angiogenesis
Further provided is a method for treating a disease associated with pathological angiogenesis in a subject, comprising contacting an EphB receptor-expressing cell of the subject which promotes angiogenesis upon binding a tetrameric EphB receptor-binding ligand with a substance which prevents binding of the tetrameric ligand, thereby disrupting angiogenesis and treating a disease associated with pathological angiogenesis.
The present invention also provides a method for treating a condition associated with interruption of angiogenic processes in a subject, comprising contacting an EphB receptor-expressing cell of the subject with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting angiogenesis and treating a condition associated with interruption of angiogenic processes.
The present invention additionally provides a method for screening a substance for the ability to inhibit angiogenesis, comprising:
a) contacting the substance with a cell expressing an EphB receptor;
b) contacting the cell of step (a) with a multimeric EphB receptor-binding ligand, which promotes angiogenesis, under conditions whereby the multimeric ligand can bind the receptor; and
c) detecting angiogenesis in cells contacted with the substance, as compared to angiogenesis in cells not contacted with the substance, whereby no angiogenesis in cells contacted with the substance and angiogenesis in cells not contacted with the substance identifies a substance having the ability to inhibit angiogenesis.
Finally, the present invention provides a method for screening an EphB-receptor binding ligand for the ability to promote angiogenesis when in multimeric form, comprising:
a) contacting a multimeric EphB receptor-binding ligand with a cell which expresses an EphB receptor under conditions whereby the ligand can bind the receptor; and
b) detecting angiogenesis of cells which have bound multimeric ligand as compared to angiogenesis of cells which have not bound multimeric ligand, whereby angiogenesis of cells which have bound multimeric ligand and no angiogenesis of cells which have not bound multimeric ligand identifies an EphB receptor-binding ligand with the ability to promote angiogenesis when in multimeric form.